This invention relates to nucleic acid and amino acid sequences of human cyclic nucleotide phosphodiesterases and their mammalian variants and to the use of these sequences in the diagnosis and treatment of cancer and immune disorders.
Cyclic nucleotides (cAMP and cGMP) function as intracellular second messengers to transduce a variety of extracellular signals including hormones, light, and neurotransmitters. Cyclic nucleotide phosphodiesterases (PDEs) degrade cyclic nucleotides to the corresponding monophosphates, thereby regulating the intracellular concentrations of cyclic nucleotides and their effects on signal transduction. At least seven families of mammalian PDEs have been identified based on substrate specificity and affinity, sensitivity to cofactors, and sensitivity to inhibitory drugs (Beavo (1995) Physiol Rev 75:725-748). Several of these families contain distinct genes, many of which are expressed in different tissues as splice variants. Within families, there are multiple isozymes and multiple splice variants of those isozymes. The existence of multiple PDE families, isozymes, and splice variants presents an opportunity for regulation of cyclic nucleotide levels and functions.
Type 1 PDEs (PDE1s) are Ca2+/calmodulin-dependent and appear to be encoded by three different genes, each having at least two different splice variants. PDE1s have been found in the lung, heart, and brain. Some of the Ca2+/calmodulin-dependent PDEs are regulated in vitro by phosphorylation and dephosphorylation. Phosphorylation of PDE1 decreases the affinity of the enzyme for calmodulin, decreases PDE activity, and increases steady state levels of cAMP. PDE2s are cGMP stimulated PDEs that are localized in the brain and are thought to mediate the effects of cAMP on catecholamine secretion. PDE3s are one of the major families of PDEs present in vascular smooth muscle. PDE3s are inhibited by cGMP, have high specificity for cAMP as a substrate, and play a role in cardiac function. One isozyme of PDE3 is regulated by one or more insulin-dependent kinases. PDE4s are the predominant isoenzymes in most inflammatory cells, and some PDE4s are activated by cAMP-dependent phosphorylation. PDE5s are thought to be cGMP specific but may also hydrolyze cAMP. High levels of PDE5s are found in most smooth muscle preparations, in platelets, and in the kidney. PDE6s play a role in vision and are regulated by light and cGMP. The PDE7 class, consisting of only one known member, is cAMP-specific and is most closely related to PDE4. PDE7 is not inhibited by rolipram, a specific inhibitor of PDE4 (Beavo, supra). PDE8 and PDE9 represent two new families of PDEs. PDE8s are cAMP specific, most closely related to PDE4, insensitive to rolipram, and sensitive to dipyridimole. PDE9s are cGMP specific and sensitive only to the PDE inhibitor, zaprinast.
PDEs are composed of a catalytic domain of about 270 amino acids, an N-terminal regulatory domain responsible for binding cofactors, and, in some cases, a C-terminal domain of unknown function. A conserved motif, HDXXHXGXXN, has been identified in the catalytic domain of all PDEs. In PDE5, an N-terminal cGMP binding domain spans about 380 amino acid residues and comprises tandem repeats of the conserved sequence motif N(R/K)XnFX3DE (McAllister-Lucas et al. (1993) J Biol Chem 268:22863-22873). The NKXnD motif has been shown by mutagenesis to be important for cGMP binding (Turko et al. (1996) J Biol Chem 271:22240-22244). PDE families display approximately 30% amino acid identity within the catalytic domain; however, isozymes within the same family typically display about 85-95% identity in this region (e.g. PDE4A vs PDE4B). Furthermore, within a family there is extensive sequence similarity ( greater than 60%) outside the catalytic domain; while across families, there is little or no sequence similarity.
Many functions of immune and inflammatory responses are inhibited by agents that increase intracellular levels of cAMP (Verghese et al. (1995) Mol Pharmacol 47:1164-1171). A variety of diseases have been attributed to increased PDE activity and associated with decreased levels of cyclic nucleotides. A form of diabetes insipidus in the mouse has been associated with increased PDE4 activity, and an increase in low-Km cAMP PDE activity has been reported in leukocytes of atopic patients. Defects in PDEs have also been associated with retinal disease. Retinal degeneration in the rd mouse, autosomal recessive retinitis pigmentosa in humans, and rod/cone dysplasia 1 in Irish Setter dogs have been attributed to mutations in the PDE6B gene. PDE3 has been associated with cardiac disease.
Many inhibitors of PDEs have been identified and have undergone clinical evaluation. PDE3 inhibitors are being developed as antithrombotic agents, antihypertensive agents, and as cardiotonic agents useful in the treatment of congestive heart failure. Rolipram, a PDE4 inhibitor, has been used in the treatment of depression, and other inhibitors of PDE4 are undergoing evaluation as anti-inflammatory agents. Rolipram has also been shown to inhibit lipopolysaccharide induced TNF-xcex1 which has been shown to enhance HIV-1 replication in vitro. Therefore, rolipram may inhibit HIV-1 replication (Angel et al. (1995) AIDS 9:1137-44). Additionally, rolipram, based on its ability to suppress the production of cytokines ,such as TNF-xcex1 and xcex2 and interferon xcex3, has been shown to be effective in the treatment of encephalomyelitis. Rolipram may also be effective in treating tardive dyskinesia and was effective in treating multiple sclerosis in an experimental animal model (Sommer et al. (1995) Nature Med 1:244-248; Sasaki et al. (1995) Eur J Pharmacol 282:71-76).
Theophylline is a nonspecific PDE inhibitor used in the treatment of bronchial asthma and other respiratory diseases. Theophylline is believed to act on airway smooth muscle function and in an anti-inflamatory or immunomodulatory capacity in the treatment of respiratory diseases (Banner and Page (1995) Eur Respir J 8:996-1000). Pentoxifylline is another nonspecific PDE inhibitor used in the treatment of intermittent claudication and diabetes-induced peripheral vascular disease. Pentoxifylline is also known to block TNF-xcex1 production and may inhibit HIV-1 replication (Angel, supra).
PDEs have also been reported to effect cellular proliferation of a variety of cell types and have been implicated in various cancers. Bang et al. (1994; Proc Natl Acad Sci 91:5330-5334) reported that growth of prostate carcinoma cell lines DU 145 and LNCaP was inhibited by delivery of cAMP derivatives and phosphodiesterase inhibitors. These cells also showed a permanent conversion in phenotype from epithelial to neuronal morphology. Others have suggested that PDE inhibitors have the potential to regulate mesangial cell proliferation and lymphocyte proliferation (Matousovic et al. (1995) J Clin Invest 96:401-410; Joulain et al. (1995) J Lipid Mediat Cell Signal 11:63-79, respectively). Finally, Deonarain et al.(1994; Br J Cancer 70:786-94) describe a cancer treatment that involves intracellular delivery of phosphodiesterases to particular cellular compartments of tumors which results in cell death.
The discovery of a new human cyclic nucleotide phosphodiesterase and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis and treatment of cancer and immune disorders.
The invention is based on the discovery of new human cyclic nucleotide phosphodiesterases referred to collectively as xe2x80x9cHSPDE10Axe2x80x9d and individually as xe2x80x9cHSPDE10A xe2x80x9d and xe2x80x9cHSPDE10A2xe2x80x9d, the polynucleotides encoding HSPDE10A1 and HSPDE10A2, and the use of these compositions for the diagnosis and treatment of cancer and immune disorders.
The invention features a purified protein comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof. The invention provides a purified variant having at least 90% amino acid sequence identity to the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof.
The invention provides an isolated polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof. The invention also provides an isolated polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof. The invention further provides an isolated polynucleotide having a sequence which is complementary to the polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof.
The invention provides an isolated polynucleotide comprising the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:4, or fragments thereof and isolated variants having at least 70% polynucleotide sequence identity to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or fragments thereof. The invention also provides an isolated polynucleotide having a sequence complementary to the polynucleotide comprising the polynucleotide sequence of SEQ ID NO:2, SEQ ID NO:4, or fragments thereof.
The invention provides a method for detecting a polynucleotide in a sample containing nucleic acids comprising hybridizing the complement of the polynucleotide to at least one of the nucleic acid of the sample, thereby forming a hybridization complex, and detecting the hybridization complex, wherein the presence of the hybridization complex indicates the presence of the polynucleotide in the sample. In one aspect, the method further comprises amplifying the nucleic acids of the sample prior to hybridization.
The invention also provides an expression vector containing the polynucleotide encoding the protein comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3. In one aspect, the expression vector is contained within a host cell. The invention further provides a method for using a polynucleotide to produce a protein comprising culturing the host cell under conditions for expression of the protein and recovering the protein from the culture. The invention further provides a composition comprising a purified protein having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof in conjunction with a pharmaceutical carrier. The invention also provides a method of using the protein to screen a plurality of molecules or compounds to identify a ligand comprising combining the protein with the molecules or compounds under conditions to allow specific binding and detecting specific binding, thereby identifying a ligand which specifically binds the protein. In one aspect, the molecules and compounds are selected from the group consisting of DNA molecules, RNA molecules, peptide nucleic acid molecules, peptides, proteins, agonists, antagonists, inhibitors, drug compounds and pharmaceutical agents. The invention further provides a composition comprising a purified agonist which specifically binds the protein having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof.
The invention provides methods for using the protein to produce polyclonal or monoclonal antibodies which specifically bind the protein. The invention also provides a purified antibody which specifically binds to a protein comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO:3. The invention further provides a method for using the antibody in an assay for adenofibromatous hyperplasia of the prostate comprising contacting the antibody with a sample from a patient, detecting complex formation between the antibody and protein, comparing complex formation with a standard, wherein the difference in complex formation indicates the presence of the disease.
The invention provides a method for treating adenofibromatous hyperplasia of the prostate as it is associated with decreased expression or activity of HSPDE10A comprising administering to a subject in need of such treatment a composition comprising a purified protein having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof, in conjunction with a pharmaceutical carrier. The invention also provides a method for treating adenofibromatous hyperplasia of the prostate comprising administering to a subject in need of such treatment a composition comprising an agonist which specifically binds the protein having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof
The invention also provides a method for treating a disorder associated with increased expression or activity of HSPDE10A, the method comprising administering to a subject in need of such treatment an antagonist of the protein having the amino acid sequence of SEQ ID NO:1, SEQ ID NO:3, or portions thereof.